Topoisomerase-based reagents and methods for molecular cloning

ABSTRACT

This invention provides a modified vaccinia topoisomerase enzyme containing an affinity tag which is capable of facilitating purification of protein-DNA complexes away from unbound DNA. This invention further provides a modified sequence specific topoisomerase enzyme.  
     This invention provides a method of ligating duplex DNAs, a method of molecular cloning of DNA, a method of synthesizing polynucleotides, and a method of gene targeting.  
     Lastly, this invention provides a recombinant DNA molecule composed of segments of DNA which have been joined ex vivo by the use of a sequence specific topoisomerase and which has the capacity to transform a suitable host cell comprising a DNA sequence encoding polypeptide activity.

[0001] This invention was made with support under Grant No. GM-46330from the National Institutes of Health, U.S. Department of Health andHuman Services. Accordingly, the United States Government has certainrights in the invention.

[0002] Throughout this application, various publications are referencedby Arabic numerals in brackets. Full citations for these publicationsmay be found at the end of the specification immediately preceding theclaims. The disclosures of these publications are in their entiretyhereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

[0003] Construction of chimaeric DNA molecules in vitro reliestraditionally on two enzymatic steps catalyzed by separate proteincomponents. Site-specific restriction endonucleases are used to generatelinear DNAs with defined termini that can then be joined covalently attheir ends via the action of DNA ligase.

[0004] Vaccinia DNA topoisomerase, a 314-aa virus-encoded eukaryotictype I topoisomerase [11], binds to duplex DNA and cleaves thephosphodiester backbone of one strand. The enzyme exhibits a high levelof sequence specificity, akin to that of a restriction endonuclease.Cleavage occurs at a consensus pentapyrimidine element5′-(C/T)CCTT^(ø)in the scissile strand [12, 5, 6]. In the cleavagereaction, bond energy is conserved via the formation of a covalentadduct between the 3′ phosphate of the incised strand and a tyrosylresidue (Tyr-274) of the protein [10]. Vaccinia topoisomerase canreligate the covalently held strand across the same bond originallycleaved (as occurs during DNA relaxation) or it can religate to aheterologous acceptor DNA and thereby create a recombinant molecule [7,8].

[0005] The repertoire of DNA joining reactions catalyzed by vacciniatopoisomerase has been studied using synthetic duplex DNA substratescontaining a single CCCTT cleavage site. When the substrate isconfigured such that the scissile bond is situated near (within 10 bpof) the 3′ end of a DNA duplex, cleavage is accompanied by spontaneousdissociation of the downstream portion of the cleaved strand [4]. Theresulting topoisomerase-DNA complex, containing a 5′ single-strandedtail, can religate to an acceptor DNA if the acceptor molecule has a 5′OH tail complementary to that of the activated donor complex. Sticky-endligation by vaccinia topoisomerase has been demonstrated using plasmidDNA acceptors with four base overhangs created by restrictionendonuclease digestion [8].

SUMMARY OF THE INVENTION

[0006] This invention provides a modified vaccinia topoisomerase enzymecontaining an affinity tag which is capable of facilitating purificationof protein-DNA complexes away from unbound DNA. This invention furtherprovides a modified sequence specific topoisomerase enzyme.

[0007] This invention provides a method of ligating duplex DNAs, amethod of molecular cloning of DNA, a method of synthesizingpolynucleotides, and a method of gene targeting.

[0008] Lastly, this invention provides a recombinant DNA moleculecomposed of segments of DNA which have been joined ex vivo by the use ofa sequence specific topoisomerase and which has the capacity totransform a suitable host cell comprising a DNA sequence encodingpolypeptide activity.

BRIEF DESCRIPTION OF THE FIGURES

[0009] FIGS. 1A-1C: Sticky-end ligation.

[0010]FIG. 1A: Topoisomerase-mediated cleavage of a 24-nucleotideCCCTT-containing hairpin substrate was assayed as a function of enzymeconcentration. The structure of the substrate is shown; the site ofstrand scission is indicated by the arrow. Reaction mixtures (20 ml)containing 50 mM Tris HCl (pH 7.5), 0.5 pmol of 5′ ³²P-labeled DNA, andtopoisomerase were incubated at 37° C. for 5 min. Covalent complexeswere trapped by addition of SDS to 1%. Samples were then electrophoresedthrough a 10% polyacrylamide gel containing 0.1% SDS. Covalent complexformation was revealed by transfer of radiolabeled DNA to thetopoisomerase polypeptide as detected by autoradiographic exposure ofthe dried gel. The extent of adduct formation was quantitated byscintillation counting of an excised gel slice containing the labeledprotein and was expressed as the percent of the input 5′ ³²P-labeledoligonucleotide that was covalently transferred to protein.

[0011]FIG. 1B: Reaction mixtures containing 50 mM Tris HCl (pH 7.5), 460pmol of 5′ ³²P-labeled hairpin substrate, and 2 pmol of topoisomerasewere incubated for 5 min at 37° C., then supplemented with linear pUC18DNAceptor (350 fmol of ends) as indicated and incubated for another 5min at room temperature. Samples were adjusted to 0.2 M NaCl and 0.5%SDS, then electrophoresed through a 1.2% agarose gel in TBE (90 mM Tris,90 mM borate, 2.5 mM EDTA) with 0.5 mg/ml ethidium bromide. DNA wasvisualized by photographing the stained gel under short wave UVillumination.

[0012]FIG. 1C: The same gel was then dried and exposed forautoradiography. The positions of the radiolabeled topoisomerase-DNA“donor” complex and the pUC strand transfer product are indicated at theright. pUC18 DNA used as acceptor in the strand transfer reactions waslinearized quantitatively by digestion with a single-cut restrictionenzyme. The 5′ phosphate termini of the linear DNAs were converted to 5′OH ends by treatment of the DNAs with calf intestinal phosphatase asindicated (CIP). The acceptor DNAs included in each reaction arespecified according to lane number. Lane M (left panel) contains DNAsize markers (1 HindIII digest).

[0013]FIG. 2: Monovalent, bivalent, and trivalent substrates.

[0014] The structure of the complementary hairpin oligonucoleotides S300and S301 are shown. The 5′ terminus is indicated by an asterisk. TheCCCTT recognition site of topoisomerase cleavage is underlined. Thestructure of the bivalent linker DNA formed by annealing S300 and S301strands is shown in the middle. At bottom is the structure of thetrivalent Y-branched linker formed by annealing S300, S304, and S303oligonucleotides.

[0015] FIGS. 3A-3C: Topoisomerase-mediated cleavage of monovalent,bivalent, and trivalent substrates.

[0016]FIG. 3A: Radiolabeled cleavage substrates were electrophoresedthrough a native polyacrylamide gel (7.5% acrylamide, 0.2%bisacrylamide) in TBE at 100 V. An autoradiogram of the dried gel isshown. Lane 1 contains the 5′ ³²P-46-mer “flip” hairpin (S300; FIG. 2).Lane 2 contains the 46-bp divalent cleavage substrate (FIG. 2). Thisstructure was formed by annealing the 5′ ³²p-S300 strand to a 3-foldmolar excess of unlabeled 46-nt complementary strand (S301, or “flop”strand; FIG. 2). Lane 3 contains the trivalent Y-branch substrate formedby annealing 5′ ³²P-S300 to two unlabeled 46-mer oligos (S303 and S304),each present at 3-fold molar excess over the labeled strand.

[0017]FIG. 3B: Cleavage reaction mixtures (20 ml) contained 50 mM TrisHCl (pH 7.5), 0.6 pmol of 5′ ³²P-labeled DNA, and 20 pmol oftopoisomerase (lanes 2, 4, 6, and 8) were incubated at 37cC for 5 min.Enzyme was omitted from control reactions (lanes 1, 3, 5, and 7).Covalent complexes were trapped by addition of SDS to 1%. (Note that thesamples were not heat-denatured). Labeled cleavage products wereresolved by SDS-PAGE. Free DNA migrated with the bromophenol blue dyefront. The structures of the various covalent protein-DNA complexes areindicated at the right of the autoradiogram. The positions and sizes (inkDa) of prestained marker proteins are indicated at the left. The inputsubstrates are illustrated at the bottom of the autoradiogram: *S300(lanes 1 and 2); *S301 (lanes 3 and 4); *S300/S301 (lanes 5 and 6);S300/*S301 (lanes 7 and 8).

[0018]FIG. 3C: Cleavage reactions contained 0.36 pmol of radiolabeledY-branch substrate (*S300/S303/S304) and 20 pmol of topoisomerase (lane2). Enzyme was omitted from a control reaction (lane 1). The structuresof the various covalent protein-DNA complexes are indicated at the rightof the autoradiogram. The positions and sizes (in kDa) of prestainedmarker proteins are indicated at the left.

[0019] FIGS. 4A-4B: Topoisomerase-mediated joining of two ends via abivalent linker.

[0020]FIG. 4A: Reaction mixtures (20 ml) contained 50 mM Tris HCl (pH7.5), 2 pmol of topoisomerase, and either 5′ ³²P-labeled monovalentsubstrate (*S300, 0.6 pmol-lanes 1 and 2) or 5′ ²P-labeled bivalentlinker (0.3 pmol of *S300/S301, i.e., 0.6 pmol of ends—lanes 3 and 4).After incubation for 5 min at 37° C., the reactions were supplementedwith 5′—OH HindIII-cut pUC18 DNA acceptor (380 fmol of ends) asindicated and incubated for another 5 min at room temperature. Sampleswere adjusted to 0.2 M NaCl and 0.5% SDS, then electrophoresed through a1.2% agarose gel in TBE. The ethidium bromide stained gel is shown atleft. The positions and sizes (kbp) of marker DNA fragments (lane M) areindicated at the left.

[0021]FIG. 4B: The same gel was dried and exposed for autoradiography.The positions of the radiolabeled topoisomerase-DNA “donor” complex andthe strand transfer products are indicated at right by arrows.

[0022] FIGS. 5A-5D: Molecular cloning of DNA using vacciniatopoisomerase.

[0023]FIG. 5A: Ligation reactions for topoisomerase-based cloning wereperformed as described under Experimental Details. The protocol isillustrated schematically.

[0024] FIGS. 5B-5C: Plasmid DNA was prepared from bacteria containingpUC18 (the parent vector, FIG. 5B) and pUC-T11 (a representativetranformant from the topoisomerase ligation reaction, FIG. 5C). DNA wasdigested with the restriction endonucleases specified above each laneusing reaction buffers provided by the vendor. Undigested plasmid DNA isshown in Lane “—”. Lane M contains DNA size markers. The positions andsizes (kbp) of reference fragments are indicated. FIG. 5D: The structureof the 46-bp bivalent linker is indicated. Diagnostic restriction siteswithin the linker are specified above the sequence.

[0025] FIGS. 6A-6B: Topoisomerase-mediated joining of two ends via atrivalent linker.

[0026]FIG. 6A: Each strand of the trivalent substrate (FIG. 2) was 5′labeled and gel-purified. The Y-branched substrate was generated byannealing equimolar amounts of the three strands (*S300, *S303, *S304).The annealed product was analyzed by electrophoresis through a native7.5% polyacrylamide gel. An autoradiograph of the gel is shown. Thetrivalent substrate is in lane 3. Component strands were analyzed inparallel (*S303 in lane 1; *S304 in lane 2) The structures of thelabeled species are indicated at the right.

[0027]FIG. 6B: Reaction mixtures (20 ml) contained 50 mM Tris HCl (pH7.5), 1 pmol of topoisomerase, and either 5′ ³²P-labeled monovalentsubstrate (*S304—lanes 1 and 2) or 5′ 3²P-labeled trivalent linker (0.3pmol of *S300/*S303/*S304—lanes 3 and 4). Each reaction contained 350fmol of input substrate (expressed as cleavable ends). After incubationfor 5 min at 37° C., the reactions were supplemented with 5′—OHHindIII-cut pUC18 DNA acceptor (570 fmol of ends) as indicated andincubated for another 5 min at room temperature. Samples were adjustedto 0.2 M NaCl and 0.5% SDS, then electrophoresed through a 1.2% agarosegel in TBE. The ethidium bromide stained gel is shown. The positions andsizes (kbp) of marker DNA fragments (lane M) are indicated at the left.

[0028]FIG. 6C: The same gel was dried and exposed for autoradiography.The positions of the radiolabeled topoisomerase-DNA “donor” complex andthe strand transfer products are indicated at right by arrows andbrackets.

[0029]FIG. 7: Expected products of bivalent end-joining.

[0030] The locations of restriction sites for HindIII (H), XmnI (X),SspI (S), and AccI (A) within the linear pUC acceptors and anticipatedligation products are indicated by arrows. The pUC DNA is denoted by asolid bar. The predicted sizes of SspI and XmnI restriction fragmentsderived from each species are listed at the left. Fragments that areexpected to contain radiolabeled linker DNA are indicated by asterisks.

[0031]FIG. 8: Expected products of trivalent end-joining.

[0032] The expected products of trivalent end joining to pUC DNA areshown in the box. Digestion with XmnI is predicted to yield fourtrivalent products, which are depicted at the right. The lengths of thepUC “arms” (in kpb) are indicated.

[0033] FIGS. 9A-9C: Restriction endonuclease digestion of end-joiningreaction products.

[0034]FIG. 9A: Reaction mixtures (20 ml) contained 50 Mm Tris Hcl (pH7.5), 1 pmol of topoisomerase, and either monovalent substrate(*S300—lanes 1 and 2), divalent linker (*S300/*301—lanes 3 an 4), ortrivalent linker (*S300/*S303/*S304—lanes 5 and 6). After incubation for5 min at 37° C., the reactions were supplemented with either 5′—OHHindIII-cut pUC19 “bivalent” DNA acceptor (600 fmol linear DNA—lanes 1,3, and 5) or 5′—OH HindIII/5′—P AccI-cut PUC19 “monovalent” acceptor(500 fmol of linear DNA—lanes 2, 4, and 6) and incubated for another 5min at room temperature. The mixtures were adjusted to recommendedrestriction conditions by addition of 10x buffer concentrate (NEB2) andthe samples were digested with SspI (10 units; New England BioLabs) for60 min at 37° C. Samples were adjusted to 0.5% SDS and electrophoresedthrough a 1.2% agarose gel in TBE. An ethidium bromide stained gel isshown. The positions and sizes (kbp) of marker DNA fragments (lane M)are indicated at the left.

[0035] FIGS. 9B-9C: Cleavage reactions containing radiolabeled bivalentlinker (lanes 1 and 2) or trivalent linker (lanes 3-5) were supplementedwith divalent pUC19 acceptor (lanes 1 and 3) or monovalent pUC19acceptor (lanes 2 and 4). A control reaction received no acceptor (lane5). The strand transfer reaction products were digested with XmnI (40units) for 2 h at 37° C., then analyzed by agarose gel electrophoresis.The ethidium bromide stained gel is shown (FIG. 9B). The positions andsizes (kbp) of marker DNA fragments (lane M) are indicated at the leftof the photograph. The same gel was dried and exposed forautoradiography (FIG. 9C). The positions of the radiolabeledtopoisomerase-DNA “donor” complex and the strand transfer products areindicated at right by arrows and brackets.

DETAILED DESCRIPTION OF THE INVENTION

[0036] This invention provides a modified vaccinia. topoisomerase enzymecontaining an affinity tag. The modified vaccinia topoisomerase enzymeis capable of facilitating purification of a vaccinia topoisomerase-DNAcomplex from unbound DNA. This invention also provides a modifiedsequence specific topoisomerase enzyme. The sequence specifictopoisomerase enzyme can be any site specific type I topoisomerase.

[0037] Topoisomerases are a class of enzymes that modify the topologicalstate of DNA via the breakage and rejoining of DNA strands. Vacciniatopoisomerase enzyme is a vaccinia virus-encoded eukaryotic type Itopoisomerase. In one embodiment vaccinia topoisomerase enzyme is a 314aa virus encoded type I topoisomerase.

[0038] In another embodiment the modified vaccinia enzyme is asite-specific type I topoisomerase. Site-specific type I topoisomerasesinclude, but are not limited to, viral topoisomerases such as pox virustopoisomerases. Examples of pox virus topoisomerases include shopefibroma virus and ORF virus. Other site specific topoisomerases areknown to those skilled in the art.

[0039] In another embodiment the affinity tag includes, but is notlimited to, the following: a glutathione-S-transferase fusion tag, amaltose binding protein tag, a histidine or poly-histidine tag.

[0040] In one embodiment the vaccinia topoisomerase-DNA complex ispurified from unbound DNA by binding the histidine taggedtopoisomerase-DNA complex to a nickel column and eluting the substratewith Imidazole.

[0041] This invention provides a duplex DNA molecule, that is, adouble-stranded DNA molecule, having at each end thereof the modifiedvaccinia topoisomerase enzyme.

[0042] Vaccinia topoisomerase binds to duplex DNA and cleaves thephosphodiester backbone of one strand while exhibiting a high level ofsequence specificity, cleaving at a consensus pentapyrimidine element5′-(C/T)CCTT↓, or related sequences, in the scissile strand. In oneembodiment the scissile bond is situated in the range of 2-12 bp fromthe 3′ end of a duplex DNA. In another embodiment cleavable complexformation by vaccinia topoisomerase requires six duplex nucleotidesupstream and two nucleotides downstream of the cleavage site. Examplesof vaccinia topoisomerase cleavable sequences include, but are notlimited to, +6/−6 duplex GCCCTTATTCCC, +8/−4 duplex TCGCCCTTATTC, +10/−2duplex TGTCGCCCTTAT, and +10/−2 duplex GTGTCGCCCTTA.

[0043] As used herein, the term donor signifies a duplex DNA whichcontains a CCCTT cleavage site within 10 bp of the 3′ end and the termacceptor signifies a duplex DNA which contains a 5′—OH terminus. Oncecovalently activated by topoisomerase the donor will only be transferredto those acceptor ends to which it can base pair.

[0044] This invention provides a method of ligating duplex DNAsemploying the modified tagged vaccinia topoisomerase. In this method ofligation the donor duplex DNA substrate is a bivalent donor duplex DNAsubstrate, that is, it contains two topoisomerase cleavage sites. Oneembodiment comprises cleaving a donor duplex DNA substrate containingsequence specific topoisomerase cleavage sites by incubating the donorduplex DNA substrate with a sequence specific topoisomerase to form atopoisomerase-bound donor duplex DNA strand and incubating thetopoisomerase-bound donor duplex DNA strand with a 5′hydroxyl-terminated compatible acceptor DNA, resulting in the ligationof the topoisomerase-bound donor duplex DNA strand to the DNA acceptorstrand.

[0045] Methods of cleaving DNA by incubation with enzymes and methods ofligating DNA by incubation are known to those skilled in the art. In oneembodiment the sequence specific topoisomerase is a vacciniatopoisomerase enzyme. In another embodiment the sequence specifictopoisomerase is a modified vaccinia topoisomerase enzyme. Inembodiments using vaccinia or modified vaccinia topoisomerase enzyme thecleavage site is an oligopyrimidine motif 5′ (C/T)CCTT↓.

[0046] In one embodiment the desired subpopulation of DNA ligationproduct is purified by introducing to the 5′ end of the donor duplex DNAan affinity label. In a preferred embodiment the affinity label is abiotin moiety and purification is performed by binding thebiotin-ligated product to streptavidin. Other purification methods areknown to those skilled in the art.

[0047] Bivalent end-joining allows the assembly of linear concatamersfrom polynucleotides with compatible ends. When the linker is designedto generate the same overhang at each cleavage site, the strand transferproducts are randomly oriented as head-to-head, head-to tail, andtail-to-tail isomers. Control of the reaction can be easily achieved byusing a bivalent linker containing different overhangs at each cleavagesite; in this way, DNA acceptors prepared with two different restrictionenzymes can be assembled in a strictly head-to-tail fashion. Theligation can be made exclusively head-to-head by combining a symmetricbivalent linker with an acceptor DNA containing asymmetric ends.

[0048] Bivalent strand transfer also results in circularization of theacceptor, a property that can be exploited for molecular cloning. Forexample, by placing the topoisomerase cleavage sites on the insert (asynthetic bivalent substrate) and cloning the cleaved DNA into a plasmidvector. This strategy is well-suited to the cloning of DNA fragmentsamplified by PCR. To clone PCR products using vaccinia topoisomerase, itis necessary to include a 10-nucleotide sequence −5′-XXXXAAGGGC- at the5′ end of the two primers used for amplification. The 5′-XXXX segmentcan correspond to any 4-base overhang that is compatible with therestriction site into which the PCR product will ultimately be cloned.The amplification procedure will generate duplex molecules containingthe sequence -GCCCTT^(ø)xxxx-3′ at both 3′ ends (where xxxx is thecomplement of XXXX). Incubation of the PCR product with topoisomerasewill result in cleavage at both termini and allow the covalentlyactivated PCR fragment to be ligated to vector DNA, essentially asdescribed in FIG. 5A.

[0049] This invention also provides a method of molecular cloning ofDNA. One embodiment comprises introducing to a donor duplex DNAsubstrate a sequence specific topoisomerase cleavage site by PCRamplifying the donor duplex DNA molecule with oligonucleotide primerscontaining the sequence specific topoisomerase cleavage site; incubatingthe donor duplex DNA with a sequence specific topoisomerase, resultingin the formation of a sequence specific topoisomerase-donor duplex DNAcomplex; incubating the sequence specific topoisomerase-donor duplex DNAcomplex with a plasmid vector with a 5′ overhang compatible to thedonor; incubating the sequence specific topoisomerase-donor duplex DNAcomplex with the plasmid vector; and transforming the plasmid vectorthat has been incubated into a host cell.

[0050] In one embodiment the sequence specific topoisomerase is avaccinia topoisomerase enzyme. In another embodiment the sequencespecific topoisomerase is a modified vaccinia topoisomerase enzyme. Inembodiments using vaccinia or modified vaccinia topoisomerase enzyme thecleavage site is an oligopyrimidine motif 5′ (C/T)CCTT↓.

[0051] PCR amplification methods are known to those skilled in the art.In one embodiment, the cloning of PCR products using vacciniatopoisomerase requires including a 10-nucleotide sequence 5′-XXXXAAGGGC-at the 5′ end of the two primers used for amplification. The 5′-XXXXsegment can correspond to any 4-base overhang compatible with therestriction site into which the PCR product will be cloned. Theamplification procedure will generate duplex molecules containing thesequence -GCCCTT^(ø)xxxx-3′ at both 3′ ends (where xxxx is thecomplement of XXXX). Incubation of the PCR product with topoisomeraseresults in cleavage at both termini and allows the covalently activatedPCR fragment to be ligated to vector DNA.

[0052] Regulatory elements required for expression include promoter orenhancer sequences to bind RNA polymerase and transcription initiationsequences for ribosome binding. For example, a bacterial expressionvector includes, but is not limited to, a promoter such as the lacpromoter and for transcription initiation the Shine-Dalgarno sequenceand the start codon AUG. Similarly, a eukaryotic expression vectorincludes, but is not limited to, a heterologous or homologous promoterfor RNA polymerase II, a downstream polyadenylation signal, the startcodon AUG, and a termination codon for detachment of the ribosome. Suchvectors may be obtained commercially or assembled from the sequencesdescribed by methods well-known in the art, for example the methodsdescribed above for constructing vectors in general.

[0053] In this invention transformation of the plasmid vector is into aprokaryotic host cell, such as a bacteria cell. In a preferredembodiment the host cell is E. coli.

[0054] Topoisomerase-based cloning has several advantages overconventional ligase-based cloning of PCR products. First, thetopoisomerase procedure circumvents any problems associated withaddition of nontemplated nucleotides by DNA polymerase at the 3′ end ofthe amplified DNA. Any nontemplated base (N) at the 3′ end of a PCRproduct destined for topoisomerase-based cloning (GCCCTT^(ø)xxxxN-3′)will dissociate spontaneously upon covalent adduct formation, and willtherefore have no impact on the ligation to vector. Second, intopoisomerase-mediated cloning, the only molecule that can possibly beligated is the covalently activated insert and the insert can only betransferred to the vector. There is no potential for in vitro covalentclosure of the vector itself, which ensures low background. There isalso no opportunity for the inserts to ligate to each other (this can beguaranteed by using 5′-phosphate-terminated PCR primers), whichprecludes cloning of concatameric repeats. Third, there is no need toconsider the sequence of the DNA being amplified in designing the PCRprimers. It is commonplace in standard cloning to introduce arestriction site into the PCR primer and to cleave the PCR products withthat restriction enzyme to facilitate joining by ligase to vector. Incases where the sequence between the primers is not already known, itbecomes problematic to choose a site for the primer that is not presentin the amplified segment. This issue becomes even more relevant as PCRmethodology advances and very long targets (10-40 kbp) are amplifiedroutinely. The issue of internal topoisomerase cleavage sites (CCCTT orrelated pentapyrimidine elements) is not a significant impediment totopoisomerase-based cloning. This is because the cleavage-religationequilibrium at internal sites strongly favors the noncovalently boundstate, and at those sites that are incised, only one strand of theduplex is nicked. Internal cleavage sites can be induced to religate byraising the salt concentration, which serves to dissociate noncovalentlybound topoisomerase and drive the reaction equilibrium to the left. Incontrast, cleavage at sites near the 3′ end is virtually quantitativeand is essentially irreversible until an acceptor DNA is provided.

[0055] Topoisomerase-based cloning strategies need not be limited tocovalent activation of the insert. By designing a plasmid polylinkersuch that CCCTT sites are situated in inverted orientation on eitherside of a restriction site, one can generate a linear vector withtopoisomerase sites at both 3′ ends. Once covalently activated bytopoisomerase, the vector “donor” can be used to clone any complementaryinsert “acceptor” (which must have 5′—OH termini), thereby precludingreligation of the vector without the insert. It is worth noting that thedonor complex formed upon cleavage by topoisomerase at a 3′ proximalsite is extremely stable. The donor molecule can be transferred nearlyquantitatively to a complementary acceptor even after many hours ofincubation of the covalent topo-DNA complex at room temperature. Indeed,the topo-linker complex can be denatured with 6 M guanidine HCl and thenrenatured spontaneously upon removal of guanidine with complete recoveryof strand transferase activity. Thus, a topoisomerase-activated vectorcan be prepared once in quantity and used as many times as needed formolecular cloning.

[0056] This invention provides a method of synthesizing polynucleotides.One embodiment comprises annealing a multiple number of duplex DNAstrands to form a branched substrate containing a sequence specifictopoisomerase cleavage site at each 3′ end; cleaving the branchedsubstrate by incubation with a sequence specific topoisomerase to form abranched topoisomerase complex; and incubating the branchedtopoisomerase complex with complementary monovalent and/or bivalent DNAacceptors. This method of polynucleotide synthesis is useful for invitro end-labelling, ligand tagging, molecular cloning.

[0057] In one embodiment the sequence specific topoisomerase is avaccinia topoisomerase enzyme. In another embodiment the sequencespecific topoisomerase is a modified vaccinia topoisomerase enzyme. Inembodiments using vaccinia or modified vaccinia topoisomerase enzyme thecleavage site is an oligopyrimidine motif 5′ (C/T)CCTT↓.

[0058] In one embodiment annealing of the duplex DNA strands isperformed by mixing the DNA strands and heating to 65° C. for 5 minutes,and then allowing the mixture to slow cool to room temperature. Oneskilled in the art knows the procedures to follow for annealing duplexDNA.

[0059] In one embodiment three duplex DNA strands are used which form atrivalent Y-branched structure. Production of a Y-branched nucleic acidby the strand transfer reaction containing the trivalent linker can bedemonstrated by diagnostic restriction digestion of the reactionproducts. The yield of Y-branched products can be optimized byeliminating residual bivalent and monovalent linkers from the substratepreparation or by ensuring that all trivalent linkers were saturatedwith three bound topoisomerase molecules. Both conditions can be met, bygel-purifying the linker and by purifying the tri-covalently activatedspecies by sedimentation. As with bivalent ligation, the orientation ofthe Y-branched products can be controlled by manipulating the design ofthe linker, or by using asymmetric acceptors. Any head-to-head-to-headtype Y-branched product of trivalent strand transfer can, in theory, beorganized into a trivalent lattice by adding a second trivalent donorcomplex that is complementary to the “tail” of the original acceptorDNA. Donor substrates of higher order valence can be used to achievetopo-based synthesis of three dimensional lattices and polyhedra fromDNA. Topoisomerase-based synthesis offers a potentially powerfulalternative strategy for building complex biopolymers.

[0060] In one embodiment a duplex DNA strand is 5′ labeled and the 5′labeled duplex DNA strand is annealed to the two duplex DNA strands toenable radiochemical purification of the substrate. Methods ofradiochemical purification are known to those skilled in the art.

[0061] This invention provides a method of gene targeting. Genetargeting involves the introduction of DNA into a cell. The DNA is takenup into the chromosomal DNA by virtue of a topoisomerase-bound donorduplex DNA. The bound topoisomerase seals the donor DNA to chromosomalDNA. One embodiment comprises cleaving a bivalent donor duplex DNAsubstrate containing a sequence specific topoisomerase cleavage site byincubating the donor duplex DNA substrate with a sequence specifictopoisomerase to form a topoisomerase-bound donor duplex DNA strand; andtransfecting the topoisomerase-bound donor duplex DNA to a suitablecell.

[0062] In one embodiment the sequence specific topoisomerase is avaccinia topoisomerase enzyme. In another embodiment the sequencespecific topoisomerase is a modified vaccinia topoisomerase enzyme. Inembodiments using vaccinia or modified vaccinia topoisomerase enzyme thecleavage site is an oligopyrimidine motif 5′ (C/T)CCTT↓.

[0063] Transfection may be performed by any of the standard methodsknown to one skilled in the art, including, but not limited toelectroporation, calcium phosphate transfection or lipofection.

[0064] This invention provides a recombinant DNA molecule composed ofsegments of DNA which have been joined ex vivo or in vitro by the use ofa sequence specific topoisomerase and which has the capacity totransform a suitable host cell comprising a DNA sequence encodingpolypeptide activity.

[0065] In one embodiment the sequence specific topoisomerase is avaccinia topoisomerase enzyme. In another embodiment the sequencespecific topoisomerase is a modified vaccinia topoisomerase enzyme. Inembodiments using vaccinia or modified vaccinia topoisomerase enzyme thecleavage site is an oligopyrimidine motif 5′ (C/T)CCTT↓.

[0066] This invention is further illustrated in the Experimental Detailssection which follows. This section is set forth to aid in anunderstanding of the invention but is not intended to, and should not beconstrued to, limit in any way the invention as set forth in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS:

[0067] I. Methods

[0068] A) Enzyme Purification:

[0069] Vaccinia DNA topoisomerase was expressed in Escherichia coli andpurified as described [9]. The heparin agarose enzyme fraction used inthe present study was the same preparation described previously [9]. Theenzyme was nearly homogeneous with respect to the 33 kDa topoisomerasepolypeptide, as determined by SDS-polyacrylamide gel electrophoresis.Protein concentration was determined using the Biorad dye reagent,taking bovine serum albumin as the standard.

[0070] B) Synthesis of 5′ Labeled Oligonucleotide Substrates:

[0071] Synthesis of DNA oligonucleotides via DMT-cyanoethylphosphoramidite chemistry was performed by the Sloan-KetteringMicrochemistry Laboratory using an Applied Biosystems model 380B ormodel 394 automated DNA synthesizer according to protocols specified bythe manufacturer. Oligonucleotides containing the CCCTT cleavage motifwere labeled at the 5′ end via enzymatic phosphorylation in the presenceof [g³²P]ATP and T4 polynucleotide kinase. Reaction mixtures (25 ml)typically contained 50 mM Tris HCl (pH 8.0), 10 mM dithiothreitol, 10 mMMgCl₂, 0.1 mM ATP, 100 mCi [g³²P]ATP, T4 polynucleotide kinase (20units, Bethesda Research Laboratories), and 500 pmol of DNAoligonucleotide (DNA was quantitated by A₂₆₀). Incubation was for 60 minat 37° C. Labeled DNA was freed of protein and radioactive nucleotide byelectrophoresis through a non-denaturing 18%. polyacrylamide gel.Full-sized labeled oligonucleotlde was localized by autoradiographicexposure of the wet gel and the labeled DNA was recovered from anexcised gel slice by soaking the slice in 0.4 ml H₂O for 8 h at roomtemperature. Hybridization of labeled DNAs to complementaryoligonucleotides was performed in 0.2 M NaCl by heating to 75° C.followed by slow cooling to room temperature. Annealed substrates werestored at 4° C.

[0072] C) Topoisomerase-based Cloning:

[0073] Reaction mixtures containing 50 mM Tris HCl (pH 7.5), 2 pmol oftopoisomerase, and either monovalent linker (0.6 pmol) or bivalentlinker (0.3 pmol) were incubated for 5 min at 37° C. A control reactioncontained topoisomerase but no DNA substrate. Each mixture was thensupplemented with 5′—OH HindIII-cut pUC18 DNA acceptor (380 fmol ofends) and incubated for another 5 min at room temperature. An aliquot (1ml) of each sample was used to transform E. coli DH5a using a BioRadGene Pulser electroporation apparatus. Preparation of bacterial cellsand electrotransformation were carried out as prescribed by themanufacturer. Aliquots of transformed bacteria were plated on LB agarcontaining 0.1 mg/ml ampicillin.

II. EXAMPLE 1. Sticky end Ligation:

[0074] The vaccinia topoisomerase was capable of sticky-end ligation ofduplex DNAs containing only 2 bases of potential complementarity, asshown in FIG. 1. In this experiment, the “donor” was a 24-mer hairpinoligonucleotide containing a single CCCTT motif (a “monovalent”substrate) with the scissile bond located 2 bases from the 3′ blunt end(FIG. 1A). The extent of cleavage of this substrate was proportional toenzyme concentration (FIG. 1A). The topoisomerase-DNA complex migratedas a discrete species during native agarose gel electrophoresis (FIG.1C). Addition of unlabeled 5′ hydroxyl-terminated CpG tailed linearpUC18 DNA (generated by digestion of pUC DNA with AccI followed bytreatment with alkaline phosphatase) resulted in transfer of thetopoisomerase-bound DNA strand to the linear DNA “acceptor.” The productof the strand transfer reaction was a radiolabeled 2.7 kbp linear formcontaining a hairpin end (FIG. 1C, lane 2). AccI-restricted plasmid DNAcontaining a 5′-phosphate terminus was inert as an acceptor (FIG. 1C,lane 3). [The requirement for a 5′OH-terminated acceptor excluded thepossibility that the reaction products might be formed by a conventionalDNA ligase contaminating the topoisomerase preparation]. Linear plasmidDNA containing non-complementary 5′—OH overhangs generated byrestriction with EcoRI (5′-AATT) or HindIII (5′-AGCT) were ineffectiveas acceptors (FIG. 1C, lanes 4 and 6), as was 5′—OH blunt-ended linearDNA generated by restriction with SmaI (lane 5).

III. EXAMPLE 2. Divalent Linkers as Donors:

[0075] Two 46-mer DNA strands were annealed to form a “divalent” 46-bpsubstrate containing a topoisomerase cleavage site 4 nucleotides fromeach 3′ end (FIG. 2). Successful annealing of the constituent strandswas evinced by the reduced mobility of the duplex molecule during nativegel electrophoresis (FIG. 3A, lane 2) compared to that of the hairpinDNA (FIG. 3A, lane 1). Either the “flip” or “flop” monovalent hairpinswere readily cleaved by vaccinia topoisomerase, resulting in theformation of a covalent protein-DNA adduct which migrated at 43 kDaduring SDS-PAGE (FIG. 3B, lanes 2 and 4). Incubation of topoisomerasewith the divalent duplex substrate yielded two complexes of 46 kDa and72 kDa; the 46 kDa species represents a single molecule of topoisomerasebound covalently at one of the CCCTT cleavage sites; the 72 kDa complexarises by cleavage at both sites on the same DNA molecule (FIG. 3B,lanes 6 and 8).

[0076] The monovalent hairpin DNA was transferred virtuallyquantitatively to linear pUC DNA containing a complementary 5′—OH-AGCToverhang (FIGS. 4A-4B, lane 2). Incubation of the bivalenttopoisomerase-DNA complex with the same acceptor yielded a complex setof products arising from ligation of the bivalent linker to twocomplementary ends of the linear pUC acceptor (FIGS. 4A-4B, lane 4).These included circular pUC and linear pUC concatamers. A significantfraction of the pUC acceptor molecules were subject to bivalentend-joining, as reflected in the distribution of EtBr-stained DNAproducts (FIG. 4A, lane 4). All ligation events were via theradiolabeled linker DNA, which became incorporated into the reactionproducts (FIG. 4B, lane 4).

[0077] IV. EXAMPLE 3.

Molecular Cloning of DNA Using Vaccinia Topoisomerase:

[0078] The ability of topoisomerase to join both ends of a linear DNA toa complementary acceptor suggested an alternative approach to molecularcloning. In the scheme shown in FIG. 5, the “insert” was a bivalent 46bp linker containing CCCTT sites at both 3′ ends. The sequence of thelinker included restriction sites for endonucleases NdeI, BglII, andEcoRV. Cleavage of the bivalent linker by topoisomerase generated a4-base overhang complementary to a HindIII restriction site. The“vector” was pUC DNA that had been cleaved with HindIII anddephosphorylated with alkaline phosphatase.

[0079] Addition of the vector to the bivalent topoisomerase-DNA donorcomplex should result in covalent joining of the insert to the vector.Upon transformation into E. coli, those molecules that had beencircularized should be able to give rise to ampicillin-resistantcolonies. It was found that the yield of ampicillin-resistant coloniesfrom bacteria transformed with a topoisomerase reaction mixturecontaining linear pUC and the bivalent linker was 110-fold higher thanthat observed for bacteria transformed with control topoisomerasereactions containing linear pUC and either monovalent linker or nolinker.

[0080] Plasmid DNA was recovered from cultures of six individualtransformants and analyzed by restriction endonuclease digestion inparallel with pUC18 plasmid DNA (FIG. 5B). [The restriction pattern forthe recombinant clone pUC-T11 shown in FIG. 5C was indistinguishablefrom that of the five other clones, which are not shown]. Whereas thestarting pUC18 plasmid contains no sites for EcoRV and BglII, therecombinant clone contains a single site for each enzyme, attributableto the insertion of the bivalent linker, which contains theserestriction sites. Similarly, the starting plasmid contains a singleNdeI site, whereas the recombinant clone contains a second NdeI site inthe linker insert. The size of the novel NdeI fragment in pUC-T11indicated that the linker DNA was inserted within the pUC polylinker asexpected. This was confirmed by the finding that the recombinant plasmidhad lost the original HindIII site upon strand transfer by topoisomeraseto the HindIII overhang (the strand transfer reaction should generatethe sequences AAGCTA and TAGCTT at the plasmid-insert junctions, whichwould not be cut by HindIII). The restriction site for SphI, which islocated immediately next to the HindIII site in the polylinker, wasretained in all recombinant clones (not shown), indicating that loss ofthe HindIII site was not caused by deletions occurring during strandtransfer. Thus, the bivalent linker DNA was successfully cloned into thepUC18 vector in a simple procedure that—exclusive of the bacterialtransformation step—takes only 10 minutes to execute.

V. EXAMPLE 4 Trivalent Linkers as Donors:

[0081] Three 46-mer DNA strands were annealed to form a “trivalent”Y-branched substrate containing a topoisomerase cleavage site 4nucleotides from each 3′ end (FIG. 2). To optimize radiochemical purityof the substrate, one of the strands was 5′ radiolabeled and annealed tothe two other strands, which were present in molar excess (FIG. 3A). Theradiolabeled Y-branched substrate migrated more slowly than a 46-bplinear duplex molecule during native gel electrophoresis (FIG. 3A, lane3). Anomalous electrophoretic behavior of the Y molecule was alsoevident during SDS-PAGE, where the trivalent substrate migrated at aposition equivalent to a 39 kDa protein (FIG. 3C, lane 1). The Y-branchstructure was cleaved quantitatively upon incubation with topoisomerase;three complexes were resolved, corresponding to Y-molecules with one,two, or three covalent bound topo polypeptides (FIG. 3C). Most of thecleaved DNAs contained two or three bound topoisomerase molecules.

[0082] To test strand transfer by the trivalent donor complex, theY-branched molecule was prepared by annealing equimolar amounts of theconstituent strands, each of which was radiolabeled. Although thethree-strand Y-form constituted the predominant product of the annealingreaction (FIG. 6A, lane 3), bivalent linkers were present as well (thesemolecules contain an unpaired “bubble” as indicated in FIG. 6). Theradiolabeled substrate was transferred quantitatively from thetopoisomerase-DNA donor complex to a linear pUC18 acceptor containing acomplementary 5′—OH-AGCT overhang (FIG. 6C, compare lanes 3 and 4). Acomplex array of multivalent ligation products was apparent byEtBr-staining and by autoradiography (FIGS. 6B-6C, lane 4). Theseincluded circular pUC and linear pUC concatamers as well as higher orderstructures (the species indicated by the bracket in FIG. 6C). None ofthe concatamers or higher order forms were observed in a control strandtransfer reaction containing a monovalent DNA linker (Fiqs. 6B-6C, lane2).

VI. EXAMPLE 5 Characterization of the Trivalent Strand TransferProducts:

[0083] The recombinant molecules generated by topoisomerase-mediatedend-joining were analyzed further by digestion with restrictionendonucleases that cleave once within the pUC sequence. In FIG. 7, theanticipated products of bivalent end-joining by topoisomerase are shown,along with the restriction fragments expected for each product upondigestion with SspI and XmnI. The products of trivalent end-joining areillustrated in FIG. 8. Experimental results showing the spectrum ofstrand transfer products after digestion with SspI and XmnI are shown inFIG. 9. In this analysis, each linker, which upon cleavage generated atailed donor complex compatible with a HindIII restriction site, wastested with two acceptor molecules, one bivalent and one monovalent. Thebivalent acceptor was linear pUC19 containing 5′—OH HindIII overhangs onboth ends. Strand transfer of a polyvalent linker to the bivalentacceptor allows for the formation of circular and linear concatamers ina head-to-head, tail-to-tail, or head-to tail fashion, as shown in FIG.7. The monovalent acceptor was pUC19 containing a 5′—OH HindIII site atone end and a 5′-phosphate AccI site at the other end. Transfer of thelinker by topoisomerase to the AccI terminus is precluded completely ontwo grounds; first, because the ends are not complementary and second,because topoisomerase cannot religate to a 5′-phosphate strand. Amonovalent acceptor will react with the topoisomerase donor complex atavailable compatible termini, but will not be able to form circles orconcatameric arrays. The structures of the various species can thus beinferred by direct comparison of the restriction digests from reactionin which monovalent, bivalent, and trivalent linkers were reacted withmonovalent and bivalent acceptors.

[0084] Consider the SspI digests of topoisomerase strand transferproducts in FIG. 9A. The monovalent linker was joined to either end ofthe bivalent pUC19 acceptor, but could not support circularization ordimerization. Hence the products were cleaved by SspI to yield twofragments derived from linear monomers (FIG. 9A, lane 1) (see FIG. 7).Ligation of the bivalent linker to bivalent acceptor yielded threeadditional products, a 4.1 kbp fragment diagnostic of head-to-headmultimer formation, a 1.3 kbp fragment indicative of tail-to-tailligation, and a 2.7 kbp species that derived from a circular molecule(FIG. 9A, lane 3). Ligation of the bivalent linker to a monovalentacceptor yielded the 4.1 kbp head-to-head fragment, but no fragmentsindicative of tail-to-tail or circular products (FIG. 9A, compare lanes3 and 4). This was precisely as expected, because the AccI “tail” wasinert for strand transfer. Reactions containing the trivalent Y-linkerand bivalent acceptor yielded two novel high molecular weight productsnot observed for the bivalent linker (FIG. 9A, lane 5). The largestproduct (indicated by the arrowhead in FIG. 9A), which was also observedwith trivalent linker and monovalent acceptor (FIG. 9A, lane 6), mustcorrespond to a Y-branched recombinant containing three pUC moleculesligated in head-to head fashion. The length of each arm is predicted tobe 2 kbp. The electrophoretic mobility of this species was anomalouslyslow, as expected for a branched DNA. The higher order complex unique tothe bivalent acceptor was presumed to be a Y-branched product containingpUC19 DNA ligated in a mixed head-head and head-tail orientation.

[0085] Digestion of the strand transfer products with XmnI confirmed andextended these findings (FIGS. 9B-9C) The digest of a reactioncontaining labeled bivalent linker and unlabeled bivalent pUC acceptoryielded diagnostic linear fragments of 3.7 kbp (head-to-head multimer),1.7 kbp (tail-to-tail multimer) and 2.7 kbp (circle). These productswere detected by EtBrstaining and by autoradiography (FIG. 9B, lanes 1).The 1.7 kbp species indicative of tail-to-tail ligation migrated justahead of a 1.85 fragment (derived either from end-tagged linear monomersor from head-to-tail multimers). The 1.7 kbp species was absent from thedigest of products formed with the monovalent pUC acceptor (FIG. 9B,lanes 2). Similarly, the 2.7 kbp species and the radiolabeled 0.8 kbpfragment (diagnostic of ligation to the “tail” end of pUC) were absentfrom the monovalent acceptor digest (FIG. 9B, lane 2).

[0086] The XmnI digest of products formed with labeled trivalent linkerand bivalent pUC19 acceptor contained four unique species not seen withthe bivalent linker (FIG. 9B, compare lanes 3 and 1) Three of thesemolecules were readily apparent as high molecular weight EtBr-stainedbands. The fourth species migrated barely in advance of the head-to-headlinear fragment and was best appreciated in the autoradiograph (FIG. 9C,lane 3). These molecules correspond to the four possible Y-branchstructures shown in Fig. 8. A priori, if there was no bias in ligationorientation, one would expect a 1:3:3:1 distribution of head-head-head,head-head-tail, head-tail-tail, and tail-tail-tail isomers. Indeed, thisis what was observed experimentally (FIG. 9B, lane 3). Consistent withthe predicted structures of the Y-branched products, only the largestspecies (head-head-head) was detected in the reaction of trivalentlinker with monovalent pUC acceptor.

REFERENCES

[0087] 1. Chen, J., and Seeman, N. C. (1991) Nature 350: 631-633.

[0088] 2. Cheng, S., et al. (1994) Proc. Natl. Acad. Sci. USA 91:5695-5699.

[0089] 3. Clark, J. M. (1988) Nucleic Acids Res. 16: 9677-9686.

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[0091] 5. Shuman, S. (1991a) J. Biol. Chem. 266: 1796-1803.

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[0093] 7. Shuman, S. (1992a) J. Biol. Chem. 267: 8620-8627.

[0094] 8. Shuman, S. (1992b) J. Biol. Chem. 267: 16755-16758.

[0095] 9. Shuman, S., et al. (1988) J. Biol. Chem. 263: 16401-16407.

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What is claimed is:
 1. A modified vaccinia topoisomerase enzymecontaining an affinity tag.
 2. The composition of claim 1, wherein themodified vaccinia topoisomerase enzyme is capable of facilitatingpurification of a modified vaccinia topoisomerase-DNA complex fromunbound DNA.
 3. The composition of claim 1, wherein the affinity tag isa histidine tag.
 4. A duplex DNA molecule having at each end thereof amodified topoisomerase enzyme of claim
 1. 5. The modified vacciniatopoisomerase enzyme of claim 1, wherein the modified vacciniatopoisomerase enzyme is a sequence specific topoisomerase.
 6. A methodof ligating duplex DNAs comprising: a) cleaving a bivalent donor duplexDNA substrate containing a sequence specific topoisomerase cleavagesite, by incubating the donor duplex DNA substrate with a sequencespecific topoisomerase to form a topoisomerase-bound donor duplex DNAstrand; and b) incubating the topoisomerase-bound donor duplex DNAstrand with a 5′ hydroxyl-terminated compatible acceptor I DNA,resulting in the ligation of the topoisomerase-bound donor duplex DNAstrand and the DNA acceptor strand.
 7. The method of claim 6, whereinthe sequence specific topoisomerase is a vaccinia topoisomerase enzyme.8. The method of claim 6, wherein the sequence specific topoisomerase isa modified vaccinia topoisomerase enzyme.
 9. The method of claim 6,wherein the sequence is a CCCTT cleavage site.
 10. The method of claim6, wherein the CCCTT cleavage site is within about 10 bp of the 3′ endof the DNA duplex.
 11. The method of claim 6, further comprising,purifying the desired subpopulation of DNA molecules by introducing abiotin moiety
 12. The method of claim 11, further comprising, bindingthe ligated product to streptavidin.
 13. The method of claim 6, whereinthe 5′ end of the donor duplex DNA contains an affinity label.
 14. Themethod of claim 13, wherein the affinity label is a biotin moiety.
 15. Amethod of molecular cloning DNA comprising: a) introducing to a donorduplex DNA substrate a sequence specific topoisomerase cleavage site byPCR amplifying the donor duplex DNA molecule with oligonucleotideprimers containing the cleavage site; b) incubating the donor duplex DNAwith a sequence specific topoisomerase, resulting in the formation of asequence specific topoisomerase-donor duplex DNA complex; c) incubatingthe sequence specific topoisomerase-donor duplex DNA complex with aplasmid vector with a 5′ overhang compatible to the donor; d) incubatingthe sequence specific topoisomerase-donor duplex DNA complex with theplasmid vector; and e) transforming the plasmid vector of step (c) intoa host cell.
 16. The method of claim 15, further comprising the donorduplex DNA of step (a) wherein the donor duplex DNA contains sequencespecific topoisomerase cleavage sites at both 3′ ends.
 17. The method ofclaim 15, further comprising the plasmid of step (c), wherein theplasmid vector contains the sequence specific topoisomerase cleavagesites situated in inverted orientation on either side of a restrictionenzyme cleavage site.
 18. The method of claim 15, wherein the sequencespecific topoisomerase is a vaccinia topoisomerase enzyme.
 19. Themethod of claim 15, wherein the sequence specific topoisomerase is amodified vaccinia topoisomerase enzyme.
 20. The method of claim 15,wherein the sequence is a CCCTT cleavage site.
 21. The method of claim15, wherein the CCCTT cleavage site is within about 10 bp of the 3′ endof the DNA duplex.
 22. A method of synthesizing polynucleotidescomprising: a) annealing a multiple number of duplex DNA strands to forma branched substrate containing a sequence specific topoisomerasecleavage site at each 3′ end; b) cleaving the branched substrate byincubation with a sequence specific topoisomerase to form a branchedtopoisomerase complex; and c) incubating the branched topoisomerasecomplex with complementary monovalent and/or bivalent DNA acceptors. 23.The method of claim 22, wherein three duplex DNA strands are used. 24.The method of claim 23, wherein the three duplex DNA strands form atrivalent Y-branched structure.
 25. The method of claim 24, furthercomprising, eliminating residual bivalent and/or monovalent linkers fromthe Y-branched substrate by gel purifying the substrate.
 26. The methodof claim 25, further comprising, purifying the polynucleotide of step(b) by sedimentation.
 27. The method of claim 22, wherein the sequencespecific topoisomerase is a vaccinia topoisomerase enzyme.
 28. Themethod of claim 22, wherein the sequence specific topoisomerase is amodified vaccinia topoisomerase enzyme.
 29. The method of claim 22,wherein the sequence is a CCCTT cleavage site.
 30. The method of claim22, wherein the CCCTT cleavage site is within about 10 bp of the 3′ endof the DNA duplex.
 31. The method of claim 24, wherein a duplex DNAstrand is 5′ labeled.
 32. The method of claim 31, further comprisingannealing the 5′ labeled duplex DNA strand to the two duplex DNA strandsin order to radiochemically purify the substrate.
 33. A method of genetargeting which comprises: a) cleaving a bivalent donor duplex DNAsubstrate containing a sequence specific topoisomerase cleavage site, byincubating the donor duplex DNA substrate with a sequence specifictopoisomerase to form a topoisomerase-bound donor duplex DNA strand; andb) transfecting the topoisomerase-bound donor duplex DNA to a suitablecell.
 34. The method of claim 32, wherein the sequence specifictopoisomerase is a vaccinia topoisomerase enzyme.
 35. The method ofclaim 33, wherein the sequence specific topoisomerase is a modifiedvaccinia topoisomerase enzyme.
 36. The method of claim 33, wherein thesequence is a CCCTT cleavage site.
 37. The method of claim 33, whereinthe CCCTT cleavage site is within about 10 bp of the 3′ end of the DNAduplex.
 38. A recombinant DNA molecule composed of segments of DNA whichhave been joined ex vivo or in vitro by the use of a sequence specifictopoisomerase and which has the capacity to transform a suitable hostcell comprising a DNA sequence encoding polypeptide activity.